Our goal is to purify and characterize adenosine receptors. As a preliminary step toward achieving this goal, we have developed a series of agonist and antagonist compounds in three main classes: 1) radioligands; 2) photoaffinity probes; and 3) immobilized ligands for affinity chromatography. We have developed methods for solubilizing receptors and assaying receptors after solubilization. We have identified the most useful of these six categories of compounds and we have found them to be superior to other compounds in their respective classes. We have successfully photo-affinity labeled adenosine A1 receptors from several species and tissues using both agonist and antagonist probes. A careful study of the factors necessary to optimize binding of agonist and antagonist radioligands has revealed that there are several states of the adenosine A1 receptor, only one of which binds agonists with high affinity. Manipulation of these states of the receptor, and the availability of novel 125I-labeled antagonists, should facilitate receptor purification. We plan to: 1) Further characterize adenosine receptors in the heart, brain and adipose tissue using a combination of radioligand binding to membranes or intact cells, photoaffinity labeling, and functional assays we have developed based on the ability of adenosine agonists and antagonists to influence the contractility of cardiac muscle and cyclic AMP content of heart cells, brain cells and adipocytes; 2) Purify adenosine receptors to homogeneity using combinations of immobilized ligand affinity chromatography, lectin affinity chromatography, immunoaffinity chromatography, high pressure gel filtration or anion exchange chromatography. When we successfully purify adenosine receptors, we will attempt to raise antibodies directed against them, determine the amino acid sequence near the radioligand binding domain, and use receptors for reconstitution experiments. 3) In the long-term, we plan to use adenosine receptor antibodies and fluorescently tagged ligands to study adenosine-receptor subcellular processing. The proposed studies will clarify the fundamental nature of adenosine receptor-effector coupling and differences in adenosine receptor binding characteristics in various tissues and may facilitate the development of tissue-specific therapeutic agents. Possible therapeutic uses for adenosine agonists or antagonists include cardiac arrhythmias, hypertension, asthma, obesity, sleep disorders and diabetes.